CD70–CD27 ligation between neural stem cells and CD4+ T cells induces Fas–FasL-mediated T-cell death

1. Introduction : Neural stem cells (NSCs) are among the most promising candidates for cell replacement therapy in neuronal injury and neurodegenerative diseases. One of the remaining obstacles for NSC therapy is to overcome the alloimmune response on NSCs by the host. 2. Methods : To investigate the mechanisms of immune modulatory function derived from the interaction of human NSCs with allogeneic T cells, we examined the immune regulatory effects of human NSCs on allogeneic T cells in vitro. 3. Results : Significantly, NSCs induced apoptosis of allogeneic T cells, in particular CD4+ T cells. Interaction of CD70 on NSCs and CD27 on CD4+ T cells mediated apoptosis of T cells. Thus, blocking CD70–CD27 interaction prevented NSC-mediated death of CD4+ T cells. 4. Conclusions : We present a rational explanation of NSC-induced immune escape in two consecutive stages. First, CD70 constitutively expressed on NSCs engaged CD27 on CD4+ T cells, which induced Fas ligand expression on CD4+ T cells. Second, CD4+ T-cell apoptosis was followed by Fas–Fas ligand interaction in the CD4+ T cells.This work was supported by the Ministry of the Knowledge Economy (grants 2009-67-10033838) and a grant from Hanwha Chemical Corporation (Project No. 0411–20070011).Peer Reviewe

Molecular cloning, expression and functional characterization of miniature swine CD86

CD86 is one of the key molecules involved in the co-stimulation of T cells. The complete cDNA encoding CD86 molecule of miniature swine was cloned and analyzed. A comparison of two CD86 amino acid sequences of miniature swine and domestic swine showed only three amino acid differences suggesting that it is unlikely to affect the major structural features of the miniature swine CD86 (msCD86). In the expression study, constitutive expression of CD86 mRNA was detected in various tissues, and the aberrant expression of the transcriptional variant (putative soluble form) was noted. The cDNA and amino acid sequences for this variant were determined and compared with those for the human soluble CD86, which was previously reported to co-stimulate the T cells. Interestingly, an alignment of the two sequences revealed that 51 amino acids corresponding to the sequence for the boundary of the extracellular and intracellular domains including the transmembrane domain are deleted at almost an identical location within the full form of CD86 from both species. This suggests the possibility of a co-stimulatory function of the putative soluble msCD86. In order to determine if the cloned msCD86 molecules has co-stimulatory activity, the proliferative responses of the human CD4(+) T cells to the msCD86-transfected COS cells were measured in the presence of Con A. The results revealed that CD86/COS, but not the mock/COS, efficiently co-stimulated the proliferation of the Con A-stimulated CD4(+) T cells and this co-stimulatory effect was blocked by CTLA4-Ig. The structural and functional information on the miniature swine CD86 from this study will enable a further genetic manipulation of CD86 as a therapeutic strategy for controlling the xenogeneic T cell immune responses mediated by the CD86-CD28 signal pathway

Expression analysis of combinatorial genes using a bi-cistronic T2A expression system in porcine fibroblasts.

In pig-to-primate xenotransplantation, multiple transgenic pigs are required to overcome a series of transplant rejections. The generation of multiple transgenic pigs either by breeding or the introduction of several mono-cistronic vectors has been hampered by the differential expression patterns of the target genes. To achieve simultaneous expression of multiple genes, a poly-cistronic expression system using the 2A peptide derived from the Thosea asigna virus (T2A) can be considered an alternative choice. Before applying T2A expression system to pig generation, the expression patterns of multiple genes in this system should be precisely evaluated. In this study, we constructed several bi-cistronic T2A expression vectors, which combine target genes that are frequently used in the xenotransplantation field, and introduced them into porcine fibroblasts. The proteins targeted to the same or different subcellular regions were efficiently expressed without affecting the localization or expression levels of the other protein. However, when a gene with low expression efficiency was inserted into the upstream region of the T2A sequences, the expression level of the downstream gene was significantly decreased compared with the expression efficiency without the insertion. A small interfering RNA targeting one gene in this system resulted in the significant downregulation of both the target gene and the other gene, indicating that multiple genes combined into a T2A expression vector can be considered as a single gene in terms of transcription and translation. In summary, the efficient expression of a downstream gene can be achieved if the expression of the upstream gene is efficient

Roles of Toll-like Receptors on Islets in Xenogenic Islet Transplantation

Background: Stimulation of innate immunity through toll-like receptors (TLRs) interfered with tolerance induction in allogeneic transplantation. Recently, roles of TLRs have been reported in parenchymal cells such as renal tubular epithelial cells beyond antigen presenting cells. Here, we tried to elucidate the roles of TLRs on porcine islets in xenogeneic islet transplantation. Methods: After isolation, adult porcine islets were stimulated by poly I:C, lipopolysaccharide (LPS) and CpG. Induction of cytokines and chemokines in islets in response to TLR stimulation was assessed using RT-PCR and ELISA. Transmigration assays were performed in order to measure the chemotactic activity of islet culture supernatant. Impacts of TLR stimulation on both apoptosis and insulin secretion of islets were assessed using Annexin-V/7-AAD staining and glucose-induced insulin stimulation test, respectively. Procoagulant induction was also assessed using RT-PCR and tissue factor assay. Porcine islets were transplanted to renal subcapsular space of MyD88 knockout, TLR4 knockout and wild type C57BL6/J mice. TLR agonists (LPS, Poly I:C) were injected together with anti-CD154 antibodies (MR-1) after pig to mouse islet transplantation. Results: Porcine islets expressed TLRs at resting status. In vitro TLR stimulation upregulated expression of both cytokines (IL-6, type I interferon) and chemokines (MCP-1, RANTES, IP-10, IL-8). Islet culture supernatant after stimulation by TLR agonists induced transmigration of THP-1 and human peripheral blood mononuclear cells across the species barrier. However, TLR stimulation influenced neither apoptosis nor insulin secretion. Both expression and functional activity of tissue factor in islets were increased by TLR stimulation. Poly I:C treatment decreased graft survival in pig to wild type mouse islet transplantation. Moreover, LPS interfered with long-term graft survival induced by MR-1 in pig to TLR4 knockout or MyD88 knockout mouse islet transplantation. Conclusion: Stimulation of TLRs on porcine islets induced both proinflammatory and procoagulant responses and thereby interfered with long-term graft survival in xenogeneic islet transplantation.

Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs

Abstracts Background Pigs with SCID can be a useful model in regenerative medicine, xenotransplantation, and cancer cell transplantation studies. Utilizing genome editing technologies such as CRISPR/Cas9 system allows us to generate genetically engineered pigs at a higher efficiency. In this study, we report generation and phenotypic characterization of IL2RG knockout female pigs produced through combination of CRISPR/Cas9 system and SCNT. As expected, pigs lacking IL2RG presented SCID phenotype. Methods First, specific CRISPR/Cas9 systems targeting IL2RG were introduced into developing pig embryos then the embryos were transferred into surrogates. A total of six fetuses were obtained from the embryo transfer and fetal fibroblast cell lines were established. Then IL2RG knockout female cells carrying biallelic genetic modification were used as donor cells for SCNT, followed by embryo transfer. Results Three live cloned female piglets carrying biallelic mutations in IL2RG were produced. All cloned piglets completely lacked thymus and they had a significantly reduced level of mature T, B and NK cells in their blood and spleen. Conclusions Here, we generated IL2RG knockout female pigs showing phenotypic characterization of SCID. This IL2RG knockout female pigs will be a promising model for biomedical and translational research

Weak response of porcine C5a receptor towards human C5a in miniature pig endothelial cells and PMNs

BACKGROUND: The anaphylatoxin C5a is a potent inflammatory molecule generated during complement activation. Although some reports have implicated C5a in xenograft rejection, to date, the molecular compatibility between human C5a and porcine C5a receptor (C5aR) has been little studied. To examine the need for pC5aR-deficient pig in xenotransplantaion, we aimed to look at the degree of direct interaction between human C5a (recipient side) and porcine endothelial cells (PECs) and porcine polymorphonuclear neutrophils (PMNs) (donor side). METHODS: Following the treatment of human C5a to isolated porcine PMNs, transmigration of PMNs was measured by Transwell system and superoxide generation by cytochrome c reduction assay. Next, the effects of human C5a on several intracellular signaling pathways were further checked; actin cytoskeletal change was observed under a confocal microscope after staining with Alexa Fluor-546-phalloidin, intracellular calcium mobilization was measured by spectrofluorophotometer. The degree of direct effect of human C5a on porcine PMNs was compared with that in human PMNs. Finally, microarray was performed to monitor the effect of human C5a on gene expression of PEC and the expression of several candidate proteins was checked by flow cytometry. RESULTS: We found that human C5a was able to induce chemotaxis, superoxide generation, actin cytoskeletal change, and intracellular calcium mobilization in porcine PMNs. However, higher concentration of human C5a was required to stimulate porcine PMNs in comparison with activating human PMNs. The amino acid sequences of porcine C5aR with those of human C5aR showed a sequence homology of only 67%. To elucidate the effect of human C5a to PECs, microarray analysis following the treatment of PECs with human C5a was performed. These data showed that human C5a did not significantly affect gene transcription patterns in PECs. Additionally, treatment of PECs with human C5a also did not induce protein expression of several cell adhesion molecules, including vascular cell adhesion molecule-1, intercellular adhesion molecule-1, P-selectin, and E-selectin, or secretion of interleukin-8 from PECs. CONCLUSIONS: These results suggest that human C5a may play a minor role on PEC activation possibly due to molecular incompatibility across the species barrier